Substrate Profiling of Tobacco Etch Virus Protease Using a Novel Fluorescence-Assisted Whole-Cell Assay

Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp). The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y) and P1 (glutamine, Q) within the substrate peptide were confirmed as being the most important specificity determinants. In position P1′, glycine (G), serine (S), cysteine (C), alanine (A) and arginine (R) were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E) is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of proteases and their substrates

Intracellular systems for characterization and engineering of proteases and their substrates

Over the years, the view on proteases as relatively non-specific protein degradation enzymes, mainly involved in food digestion and intracellular protein turnover, has shifted and they are now recognized as key regulators of many biological processes that determine the fate of a cell. Besides their biological role, proteases have emerged as important tools in various biotechnical, industrial and medical applications. At present, there are worldwide efforts made that aim at deciphering the biological role of proteases and understanding their mechanism of action in greater detail. In addition, with the growing demand of novel protease variants adapted to specific applications, protease engineering is attracting a lot of attention. With the vision of contributing to the field of protein science, we have developed a platform for the identification of site-specific proteolysis, consisting of two intracellular genetic assays; one fluorescence-based (Paper I) and one antibiotic resistance-based (Paper IV). More specifically, the assays take advantage of genetically encoded short-lived reporter substrates that upon cleavage by a coexpressed protease confer either increased whole-cell fluorescence or antibiotic resistance to the cells in proportion to the efficiency with which the substrates are processed. Thus, the fluorescence-based assay is highly suitable for high-throughput analysis of substrate processing efficiency by flow cytometry analysis and cell sorting, while the antibiotic resistance assay can be used to monitor and identify proteolysis through (competitive) growth in selective media. By using the highly sequence specific tobacco etch virus protease (TEVp) as a model in our systems, we could show that both allowed for (i) discrimination among closely related substrate peptides (Paper I & IV) and (ii) enrichment and identification of the best performing substrate-protease combination from a background of suboptimal variants (Paper I & IV). In addition, the fluorescence-based assay was used successfully to determine the substrate specificity of TEVp by flow cytometric screening of large combinatorial substrate libraries (Paper II), and in a separate study also used as one of several methods for the characterization of different TEVp mutants engineered for improved solubility (Paper III). We believe that our assays present a new and promising path forward for high-throughput substrate profiling of proteases, directed evolution of proteases and identification of protease inhibitors, which all are areas of great biological, biotechnical and medical interest.QC 2011051

Intracellular systems for characterization and engineering of proteases and their substrates

Over the years, the view on proteases as relatively non-specific protein degradation enzymes, mainly involved in food digestion and intracellular protein turnover, has shifted and they are now recognized as key regulators of many biological processes that determine the fate of a cell. Besides their biological role, proteases have emerged as important tools in various biotechnical, industrial and medical applications. At present, there are worldwide efforts made that aim at deciphering the biological role of proteases and understanding their mechanism of action in greater detail. In addition, with the growing demand of novel protease variants adapted to specific applications, protease engineering is attracting a lot of attention. With the vision of contributing to the field of protein science, we have developed a platform for the identification of site-specific proteolysis, consisting of two intracellular genetic assays; one fluorescence-based (Paper I) and one antibiotic resistance-based (Paper IV). More specifically, the assays take advantage of genetically encoded short-lived reporter substrates that upon cleavage by a coexpressed protease confer either increased whole-cell fluorescence or antibiotic resistance to the cells in proportion to the efficiency with which the substrates are processed. Thus, the fluorescence-based assay is highly suitable for high-throughput analysis of substrate processing efficiency by flow cytometry analysis and cell sorting, while the antibiotic resistance assay can be used to monitor and identify proteolysis through (competitive) growth in selective media. By using the highly sequence specific tobacco etch virus protease (TEVp) as a model in our systems, we could show that both allowed for (i) discrimination among closely related substrate peptides (Paper I & IV) and (ii) enrichment and identification of the best performing substrate-protease combination from a background of suboptimal variants (Paper I & IV). In addition, the fluorescence-based assay was used successfully to determine the substrate specificity of TEVp by flow cytometric screening of large combinatorial substrate libraries (Paper II), and in a separate study also used as one of several methods for the characterization of different TEVp mutants engineered for improved solubility (Paper III). We believe that our assays present a new and promising path forward for high-throughput substrate profiling of proteases, directed evolution of proteases and identification of protease inhibitors, which all are areas of great biological, biotechnical and medical interest.QC 2011051

Novel Fluorescence-Assisted Whole-Cell Assay for Engineering and Characterization of Proteases and Their Substrates▿

We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's apparent activity in the Escherichia coli cytoplasm. We demonstrated that this system can reveal differences in the efficiency with which tobacco etch virus (TEV) protease processes different substrate peptides. In addition, when analyzing E. coli cells expressing TEV protease variants that differed in terms of their in vivo solubility, cells containing the most-soluble protease variant exhibited the highest fluorescence intensity. Furthermore, flow cytometry screening allowed for enrichment and subsequent identification of an optimal substrate peptide and protease variant from a large excess of cells expressing suboptimal variants (1:100,000). Two rounds of cell sorting resulted in a 69,000-fold enrichment and a 22,000-fold enrichment of the superior substrate peptide and protease variant, respectively. Our approach presents a new promising path forward for high-throughput substrate profiling of proteases, engineering of novel protease variants with desired properties (e.g., altered substrate specificity and improved solubility and activity), and identification of protease inhibitors

Towards an antibiotic resistance-based assay for protease characterization and engineering

Proteases attract a lot of interest, not only because of their involvement in many biological processes, but also as essential tools in biomedical research and industry. Here, we present a novel genetic method for identification of site-specific proteolysis. The assay utilizes plasmid-encoded reporters that upon processing by a coexpressed protease confer antibiotic resistance to cells in proportion to the cleavage efficiency. We demonstrate that cells expressing cleavable or non-cleavable reporters together with tobacco etch virus protease (TEVp), could be distinguished from each other by growth in selective media. Moreover, the growth rate proved to correlate with the substrate processing efficiency. Thus by applying competitive growth in antibiotic-containing medium, we could also show that the substrate preferred by TEVp was enriched at the expense of other less-efficient substrates. We believe that this simple methodology will facilitate protease substrate identification, and hold great promise for directed evolution of proteases towards improved and/or new functionality.QC 2011051

Towards an antibiotic resistance-based assay for protease characterization and engineering

Proteases attract a lot of interest, not only because of their involvement in many biological processes, but also as essential tools in biomedical research and industry. Here, we present a novel genetic method for identification of site-specific proteolysis. The assay utilizes plasmid-encoded reporters that upon processing by a coexpressed protease confer antibiotic resistance to cells in proportion to the cleavage efficiency. We demonstrate that cells expressing cleavable or non-cleavable reporters together with tobacco etch virus protease (TEVp), could be distinguished from each other by growth in selective media. Moreover, the growth rate proved to correlate with the substrate processing efficiency. Thus by applying competitive growth in antibiotic-containing medium, we could also show that the substrate preferred by TEVp was enriched at the expense of other less-efficient substrates. We believe that this simple methodology will facilitate protease substrate identification, and hold great promise for directed evolution of proteases towards improved and/or new functionality.QC 2011051

Construction, expression and characterization of TEV protease mutants engineered for improved solubility

In recent years, the highly sequence specific tobacco etch virus protease (TEVp) has emerged as one of the most popular and widely used reagents for removal of fusion tags from target proteins. Its use, however, has been hampered due to relatively poor solubility and inefficient expression in E. coli. Although a lot of progress has been made, there is still need for new and improved TEVp variants. Recently, two different gain-of-function TEVp mutants were described; one containing the substitutions L56V/S135G, which conferred improved solubility and activity in vitro, while the other mutant, containing the substitutions T17S/N68D/I77V, was claimed to yield more soluble protease than the wild-type (wt) protease upon overexpression in E. coli. Here, we analyzed if the L56V/S135G substitutions could promote increased solubility also in vivo, as that would be beneficial to TEVp production and had never been investigated before. We also intended to create a novel, and hopefully superior, TEVp variant with all five mutations combined (T17S/L56V/N68D/S135G/I77V) in a single protease molecule. This variant and the two parental TEVp variants as well as the wt protease, were all expressed in E. coli and characterized with respect to the expression levels, solubility and activity using several different techniques; among them, a newly developed fluorescence-assisted whole-cell assay that directly reports on the apparent protease activity in vivo. Our results show that the L56V/S135G substitutions improve the solubility not only in vitro but also in vivo, which did hold true for the activity as well. Disappointingly, the protease variant containing all five substitutions (T17S/L56V/N68D/S135G/I77V) did not show the best performance, which instead the L56V/S135G variant did. In contrast to an earlier report, we show that the substitutions T17S/N68D/I77V, did not improve the TEVp solubility. In fact, they reduced the activity, and even appeared to have a slightly negative effect on solubility, of all protease constructs in which they were present. Thus, the best current and most promising TEVp variant for future protease engineering efforts, towards improved expression properties and enhanced catalytic efficiency, are those containing the L56V/S135G substitutions.QC 2011051

Construction, expression and characterization of TEV protease mutants engineered for improved solubility

In recent years, the highly sequence specific tobacco etch virus protease (TEVp) has emerged as one of the most popular and widely used reagents for removal of fusion tags from target proteins. Its use, however, has been hampered due to relatively poor solubility and inefficient expression in E. coli. Although a lot of progress has been made, there is still need for new and improved TEVp variants. Recently, two different gain-of-function TEVp mutants were described; one containing the substitutions L56V/S135G, which conferred improved solubility and activity in vitro, while the other mutant, containing the substitutions T17S/N68D/I77V, was claimed to yield more soluble protease than the wild-type (wt) protease upon overexpression in E. coli. Here, we analyzed if the L56V/S135G substitutions could promote increased solubility also in vivo, as that would be beneficial to TEVp production and had never been investigated before. We also intended to create a novel, and hopefully superior, TEVp variant with all five mutations combined (T17S/L56V/N68D/S135G/I77V) in a single protease molecule. This variant and the two parental TEVp variants as well as the wt protease, were all expressed in E. coli and characterized with respect to the expression levels, solubility and activity using several different techniques; among them, a newly developed fluorescence-assisted whole-cell assay that directly reports on the apparent protease activity in vivo. Our results show that the L56V/S135G substitutions improve the solubility not only in vitro but also in vivo, which did hold true for the activity as well. Disappointingly, the protease variant containing all five substitutions (T17S/L56V/N68D/S135G/I77V) did not show the best performance, which instead the L56V/S135G variant did. In contrast to an earlier report, we show that the substitutions T17S/N68D/I77V, did not improve the TEVp solubility. In fact, they reduced the activity, and even appeared to have a slightly negative effect on solubility, of all protease constructs in which they were present. Thus, the best current and most promising TEVp variant for future protease engineering efforts, towards improved expression properties and enhanced catalytic efficiency, are those containing the L56V/S135G substitutions.QC 2011051

Construction, expression and characterization of TEV protease mutants engineered for improved solubility

In recent years, the highly sequence specific tobacco etch virus protease (TEVp) has emerged as one of the most popular and widely used reagents for removal of fusion tags from target proteins. Its use, however, has been hampered due to relatively poor solubility and inefficient expression in E. coli. Although a lot of progress has been made, there is still need for new and improved TEVp variants. Recently, two different gain-of-function TEVp mutants were described; one containing the substitutions L56V/S135G, which conferred improved solubility and activity in vitro, while the other mutant, containing the substitutions T17S/N68D/I77V, was claimed to yield more soluble protease than the wild-type (wt) protease upon overexpression in E. coli. Here, we analyzed if the L56V/S135G substitutions could promote increased solubility also in vivo, as that would be beneficial to TEVp production and had never been investigated before. We also intended to create a novel, and hopefully superior, TEVp variant with all five mutations combined (T17S/L56V/N68D/S135G/I77V) in a single protease molecule. This variant and the two parental TEVp variants as well as the wt protease, were all expressed in E. coli and characterized with respect to the expression levels, solubility and activity using several different techniques; among them, a newly developed fluorescence-assisted whole-cell assay that directly reports on the apparent protease activity in vivo. Our results show that the L56V/S135G substitutions improve the solubility not only in vitro but also in vivo, which did hold true for the activity as well. Disappointingly, the protease variant containing all five substitutions (T17S/L56V/N68D/S135G/I77V) did not show the best performance, which instead the L56V/S135G variant did. In contrast to an earlier report, we show that the substitutions T17S/N68D/I77V, did not improve the TEVp solubility. In fact, they reduced the activity, and even appeared to have a slightly negative effect on solubility, of all protease constructs in which they were present. Thus, the best current and most promising TEVp variant for future protease engineering efforts, towards improved expression properties and enhanced catalytic efficiency, are those containing the L56V/S135G substitutions.QC 2011051